Project Summary/Abstract Metabolic reprograming has been readily recognized as part of the hallmarks of cancer. Particularly, proline metabolism is critically important for tumor metastasis, as pyrroline-5-carboxylate reductase (P5CR), a key mitochondrial proline biosynthesis enzyme, is highly overexpressed in various metastasized tumors and to promote tumor cell growth. This suggests that the proline synthesis pathway is an attractive target for cancer treatment. Kaposi?s sarcoma-associated herpesvirus (KSHV) is one of oral herpesviruses and causes Kaposi?s sarcoma and pleural effusion lymphoma. The proposed research is directed toward investigating the molecular mechanism of KSHV-mediated regulation of proline metabolism. We have shown that KSHV K1 glycoprotein elicits intracellular signal transduction for growth transformation in a C-terminal immunoreceptor tyrosine-based activation motif (ITAM)-dependent manner. Surprisingly, we discovered (Aim 1) that the unphosphorylated K1 directly interacted with mitochondrial P5CR in an ITAM-independent manner and this interaction activated its enzymatic activity, increasing intracellular proline synthesis. Furthermore, KSHV gene screening identified that ORF75, an enzyme-deficient viral glutamine amidotransferase (vGAT), functions as a substrate recruiter by binding one of host GATs, PFAS (phosphoribosylformylglycinamidine synthetase), and vGAT/PFAS enzyme complex deamidates intracellular nucleic acid sensors, evading host innate immune response. Furthermore, our proteome-wide deamidation screen discovered (Aim 2) that the vGAT/PFAS complex effectively induced the deamidation of P5C synthase (P5CS) during KSHV lytic replication. Based on these, we hypothesize a novel viral regulation of proline metabolism where KSHV utilizes two gene products to deregulate proline synthesis pathway: K1 activates the enzymatic activity of P5CR in an ITAM- independent manner and vGAT collaborates with host PFAS to induce the deamidation of P5CS, which ultimately synergizes to enhance proline synthesis and cell growth transformation (Aim 3). The proposed studies will provide an understanding of a novel oncogenic strategy of KSHV to induce proline metabolism for virus-induced pathogenesis.